| Objective:To investigate the ruechanism of effect of the balance of CD4~+T cell subsets and related cytokines,including Thl(CD4~+IFN-γ~+),Th2(CD4~+IL-4~+),Thl7(CD4~+IL-17~+)and Treg(CD4~+CD25~+Foxp3~+),on Guillain-Barre syndrome(GBS),and to explore appropriate noninvasive enterobacteria-based treatments for GBS through B.infantis intervention,in an effort to find new therapies and provide theoretical and experimental basis for the clinical treatment of GBS.Materials and Methods:In this study,we recruited a group of patients with GBS and evaluated their GBS disability scale scores(GDSs).Moreover,we used ELISA to determine the levels of IL-17A,IFN-γ and IL-4 in the peripheral blood and cerebrospinal fluid(CSF)among the patients and healthy controls(HC).The concentration of intestinal Bifidobacterium infantis(B.infantis)was assayed by real-time PCR(RT-PCR).Then,we observed the correlation among them.In addition,we established a GBS Experimental Autoimmune Neuritis(EAN)rat model,whereas we evaluated the rats’ GDSs and injeeted them with either normal saline,low-concentration B.infantis or high-concentration B.infantis,and then observed the pathological changes in sciatic nerve demyelination and inflammatory infiltration.We also used electromyography to observe the changes in the conduction velocity and amplitude of sciatic nerves and in the latency of the sensory and action nerve potential.Moreover,we used ELISA to measure the levels of IL-17A,IFN-γ and IL-4 in peripheral blood,in order to observe the effects of B.infantis on the balance of IFN-γ,IL-17A and IL-4,clinical symptoms of paralysis,demyelination of peripheral nerves and nerve roots as well as inflammatory cell infiltration among the EAN rats.For another group of patients with GBS,we evaluated their GDSs and used flow cytometry to determine the levels of Thl(CD4~+IFN-γ~+),Th2(CD4~+IL-4~+),Th17(CD4~+IL-17~+)and Treg(CD4~+CD25~+Foxp3~+)cells of lymphocytes in the peripheral blood of the GBS patients and the HC,while using RT-PCR to detect the levels of intestinal bifidobacteria and Thl,Th2,Th17 and Treg cell-related transcription factors T-bet,GATA-3,RORγτ and Foxp3~+.Then,we observed the correlation among them.Moreover,we established a EAN rat model,whereas we infused either low-concentration B.infantis,high-concentration B.infantis or normal saline into their gastrointestinal tracts,and observed the changes in their GDSS scores and body weight.We used electron microscopy to examine the effects of these infusions on the degree of sciatic nerve myelin sheath and axonal degeneration and injury.Flow cytometry was used to measure the effect on the levels of Thl(CD4~+IFN-γ~+),Th2(CD4~+IL-4~+),Th17(CD4~+IL-17~+)and Treg(CD4~+CD25~+Foxp3~+)cells in the peripheral blood of the EAN rats.This study investigated the effect of the balance of CD4~+T cell subsets and related cytokines on GBS,with infusion of B.infantis into gastrointestinal tracts as an intervention,in combination with ELISA,flow cytometry,RT-PCR,HE staining and immunohistology,and with the fresh stool,peripheral blood monocytes and CSF of the GBS patients,B.infantis and EAN rats as study subjects.In this study,we,through B.infantis intervention,explored appropriate enterobacteria-based treatments for GBS,in an effort to find new therapies and provide theoretical and experimental basis for the clinical treatment of GBS.1.We examined the concentrations of of IL-17A,IFN-γ and IL-4 in peripheral blood plasma and CSF among 30 patients with GBS and 20 healthy controls,and found that the concentrations of plasma IL-17A and IFN-γ in the patients were significantly higher than those in the HC(p<0.01 VS p<0.05).In contrast,there was no statistically significant difference in the levels of IL-4(p>0.05).In CSF,only the level of IL-17A in the patients was significantly higher than that in the HC,and there was no significant difference in the levels of IFN-γ and IL-4(p>0.05).In contrast,the levels of intestinal B.infantis in the GBS patients were significantly lower than those in the HC(P<0.01).We next analyzed potential relationships between the levels of IL-17A and the values of GDSS in those patients,and found that the concentrations of plasma and CSF IL-17A were correlated positively with GDSS in GBS patients(r=0.5169,p=0.013;r=0.5388,p=0.005).In contrast,the concentrations of Bifidobacterium were correlated negatively with GDSS in GBS patients(r=-0.4796,p=0.0258).Further analysis revealed that the concentrations of plasma and CSF IL-17A were also negatively correlated with the concentrations of Bifidobacterium(r=-0.4366,p=0.0279;r=-0.4259,p=0.0341).Two weeks after the gastrointestinal infusion of normal saline,low-concentration B.infantis or high-concentration B.infantis,we found that the concentrations of peripheral plasma IL-17A and IFN-γ in the EAN rats were significantly lower than those in the model group(p<0.05 VS p<0.01),while there was no significant difference in the levels of IL-4(p>0.05).When compared with the model group,B.infantis administration promoted the conduction velocity and amplitude of sciatic nerves and reduced in the latency of the sensory and action nerve potential(p<0.05).Therefore,we concluded that IL-17A and IFN-y were involved in the occurrence and development of GBS/EAN,and that B.infantis administration can reduce the levels of Thl(CD4~+IFN-γ~+)and Thl7(CD4~+IL-17~+),ameliorate the symptoms of EAN and decrease pro-inflammatory cytokines in our rat model.2.To investigate the differentiation of CD4~+T cells in GBS patients,we used flow cytometry to detect the levels of lymphocytes Thl(CD4~+IFN-γ~+),Th2(CD4~+IL-4~+)and Th17(CD4~+IL-17~+)as well as Treg(CD4~+CD25~+Foxp3~+)cells in peripheral blood in 30 GBS patients and 20 HC.We found that the levels of Th2 and Thl 7 cells in the patients were significantly higher than those in the HC,while Treg cells decreased massively(p<0.05).To further verify the differentiation of CD4~+T cells,we used RT-PCR to examine the expression levels of the transcription factors of Thl,Th2,Thl7 and Treg cells,including T-bet,GATA-3,RORγτ and Foxp3~+.The results showed that the mRNA expressions of GATA-3 and RORγτ in the patients were remarkably higher than those in the HC(p<0.05),but there was no signifieant differenee in the mRNA expression levels of T-bet and Foxp3 between the patients and the HC(p>0.05).Next,we detected the levels of B.infantis in the stool samples using RT-PCR.The results revealed that the levels of B.infantis in the GBS patients were significantly lower than those in the HC(p<0.01).Further analysis demonstrated that the mRNA expression levels of GATA-3 and RORγτcorrelated negatively with the concentrations of B.infantis(r=-0.5866,p=0.0279;r=-0.4259,p=0.0341).On the other hand,there was no correlation between the mRNA expression level of Foxp3 and the concentrations of B.infantis in the GBS patients(r=0.234,p=0.057).These data would seem to suggest that the augmented levels of Th2 and Th17 and the decreased concentration of B.infantis may be associated with the development of GBS.Two weeks after gastrointestinal infusion of B.infantis with the EAN rats,we,through the examination of TEM,found that the ultrastructure of the sciatic nerves changed,with symptoms including swelling,disorganization,and separation of the myelin lamellae ameliorated.Moreover,B.infantis administration considerably promoted the conduction velocity and amplitude of sciatic nerves,and there was no difference between low and high concentrations in terms of this effect.Therefore,we concluded that B.infantis can ameliorate the symptoms of EAN in our rat model and reduce the levels of related pro-inflammatory cytokines.Two weeks after gastrointestinal infusion of B.infantis with the EAN rats,we used flow cytometry to detect the changes in the levels of Thl(CD4~+IFN-γ~+),Th2(CD4~+IL-4~+),Thl7(CD4~+IL-17~+)and Treg(CD4~+CD25~+Foxp3~+)cells in the peripheral blood of the EAN rats.We found that the levels of Th2 and Thl7 were significantly lower than those in the model group(p<0.01),while there was no substantial alteration in the level of Th1(p>0.05).However,the level of Treg cells was significantly higher than that in the model group(p<0.01).Fixrther studies revealed that there was no remarkable modification between the low and high concentrations of B.infantis.Therefore,the B.infantis administration potentially reduced the levels of Th2 and Th17,promoted the level of Treg cells and ameliorated the symptoms of EAN in the rat model.Results:l.CD4~+T cell subsets and related cytokines,including Thl,Th2,Thl 7 and Treg,are correlated with the pathogenesis and clinical symptoms of GBS/EAN.Mainly,Thl 7 cells promote the occurrence and development of GBS/EAN,while Tregs cells inhibit the occurrence and development and play a role in ameliorating GBS clinical symptoms.2.The levels of intestinal B.infantis in GBS/EAN patients were remarkably lower than those in the HC.The lower the concentration of intestinal B.infantis is,the higher the GDSS score is,which reveals that B.infantis can inhibit the occurrence and development of GBS/EAN and ameliorate the clinical symptoms of these diseases.3.After the administration of 5.infantis,the levels of Thl7 cells decreased significantly,while those of Tregs increased significantly.Moreover,the pathological changes in peripheral nerves and the clinical symptoms of GBS patients and EAN rats were significantly ameliorated.Therefore,the clinical symptoms of patients with GBS/EAN can be ameliorated by regulating the levels of CD4~+T cell subsets and related cytokines,especially the balance of Thl7/Tregs,with B.infantis.4.These findings may provide new information for further understanding the pathogenesis of GBS,and provide a new theoretical basis for discovering the early markers of this disease and for giving early intervention and treatment. |
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